Adventitious bud regeneration and Agrobacterium tumefaciens -mediated genetic transformation of Eucalyptus urophylla × E. tereticornis interspecific hybrid

A high-efficiency regeneration and genetic transformation system is indispensable for generating desirable traits in important trees such as Eucalyptus . However, lower regeneration efficiency is common for most varieties because of the recalcitrance of this genus. Here, a stable and highly efficient in vitro organogenesis protocol and Agrobacterium -mediated genetic transformation system of Eucalyptus were developed, and transgenic plants were obtained. In this protocol, the preferred explants were the top and middle stem internodes from in vitro micro-shoots of the E. urophylla × E. tereticornis hybrid. Modified Woody Plant Medium (mWPM) containing 0.025 mg·L -1 thidiazuron (TDZ) and 0.10 mg·L -1 indole-3-butyric acid (IBA) was used to induce multiple adventitious buds that allowed 85.6% shoot formation. The binary vector pBI121 carrying the neomycin phosphotransferase II ( nptII ) and β-glucuronidase ( uidA ) genes was applied for transformation. The preferred internodes were precultured for 0 to 3 d and infected with A. tumefaciens strain GV3101 grown to a bacterial density of 0.5 (OD600). Then, they were transferred to a co-culture medium supplemented with 50 μM acetosyringone (AS) and co-cultured for 2 d in the dark. The transgenic adventitious buds formed in regeneration medium, which was replaced by the same medium with a 2-wk subculture interval through kanamycin selection. Using the aforementioned method, transgenic plantlets can be obtained within 3 mo with a transformation frequency of 3.8%, which was verified by polymerase chain reaction amplification (PCR) and histochemical analysis of GUS activity. The constructed genetic transformation system will lay a foundation for mining gene functions and further molecular breeding of Eucalyptus .